Biomedical subjects

J Bolz

Publications and source records attributed to J Bolz.

At least 19 recordsLinked to original sources

PubMed2000

Spatial distributions of guidance molecules regulate chemorepulsion and chemoattraction of growth cones.

It is generally assumed that gradients of chemotropic molecules are instrumental to the wiring of the nervous system. Recently, two members of the secreted class III semaphorin protein family have been implicated as repulsive (Sema3A) and attractive (Sema3C) guidance molecules for cortical axons (). Here, we show that stabilized gradients of increasing semaphorin concentrations elicit stereotyped responses from cortical growth cones, independent of the absolute concentration and the slope of these gradients. In contrast, neither repulsive effects of Sema3A nor attractive effects of Sema3C were observed when axons were growing toward decreasing semaphorin concentrations. Thus, growth cone guidance by gradients of chemotropic molecules is robust and reproducible, because it is primarily independent of the exact dimensions of the gradients.

Carrier Proteins↗

PubMed1999

Opposing roles for neurotrophin-3 in targeting and collateral formation of distinct sets of developing cortical neurons.

Neurotrophin-3 and its receptor TrkC are expressed during the development of the mammalian cerebral cortex. To examine whether neurotrophin-3 might play a role in the elaboration of layer-specific cortical circuits, slices of layer 6 and layers 2/3 neurons were cultured in the presence of exogenously applied neurotrophin-3. Results indicate that neurotrophin-3 promotes axonal branching of layer 6 axons, which target neurotrophin-3-expressing layers in vivo, and that it inhibits branching of layers 2/3 axons, which avoid neurotrophin-3-expressing layers. Such opposing effects of neurotrophin-3 on axonal branching were also observed with embryonic cortical neurons, indicating that the response to neurotrophin-3 is specified at early developmental stages, prior to cell migration. In addition to its effects on fiber branching, axonal guidance assays also indicate that neurotrophin-3 is an attractive signal for layer 6 axons and a repellent guidance cue for layers 2/3 axons. Experiments with specific antibodies to neutralize neurotrophin-3 in cortical membranes revealed that endogenous levels of neurotrophin-3 are sufficient to regulate branching and targeting of cortical axons. These opposing effects of neurotrophin-3 on specific populations of axons demonstrate that it could serve as one of the signals for the elaboration of local cortical circuits.

Animals↗

PubMed1998

Membrane-associated molecules guide limbic and nonlimbic thalamocortical projections.

Membrane-associated signals expressed in restricted domains of the developing cerebral cortex may mediate axon target recognition during the establishment of thalamocortical projections, which form in a highly precise manner during development. To test this hypothesis, we first analyzed the outgrowth of thalamic explants from limbic and nonlimbic nuclei on membrane substrates prepared from limbic cortex and neocortex. The results show that different thalamic fiber populations are able to discriminate between membrane substrates prepared from target and nontarget cortical regions. A candidate molecule that could mediate selective choice in the thalamocortical system is the limbic system-associated membrane protein (LAMP), which is an early marker of cortical and subcortical limbic regions (Pimenta et al.,1995) that can promote outgrowth of limbic axons. Limbic thalamic and cortical axons showed preferences for recombinant LAMP (rLAMP) in a stripe assay. Incubation of cortical membranes with an antibody against LAMP prevented the ability of limbic thalamic fibers to distinguish between membranes from limbic cortex and neocortex. Strikingly, nonlimbic thalamic fibers also responded to LAMP, but in contrast to limbic thalamic fibers, rLAMP inhibited branch formation and acted as a repulsive axonal guidance signal for nonlimbic thalamic axons. The present studies indicate that LAMP fulfills a role as a selective guidance cue in the developing thalamocortical system.

Animals↗

PubMed1998

Dual action of a ligand for Eph receptor tyrosine kinases on specific populations of axons during the development of cortical circuits.

The structural basis of cortical columns are radially oriented axon collaterals that form precise connections between distinct cortical layers. During development, these connections are highly specified from the initial outgrowth of collateral branches. Our previous work provided evidence for positional cues confined to individual layers that induce and/or prevent the formation of axon collaterals in specific populations of cortical neurons. Here we demonstrated with in situ hybridization techniques that mRNA of the Eph receptor tyrosine kinase EphA5 and one of its ligands, ephrin-A5, are present in distinct cortical layers, at a time when intrinsic connections are being formed in the cortex. Axonal guidance assays indicate that ephrin-A5 is a repellent signal for a populations of axons that in vivo avoid the cortical layer expressing ephrin-A5. In contrast to its established role as a repulsive axonal guidance signal, ephrin-A5 specifically mediates sprouting of those cortical axons that target the ephrin-A5-expressing layer in vivo. These results identify a novel function of ephrin-A5 on axonal arbor formation. The laminar distribution and the dual action on specific populations of axons suggest that ephrin-A5 plays a role in the assembly of local cortical circuits.

Aging↗

PubMed1998

Semaphorins act as attractive and repulsive guidance signals during the development of cortical projections.

Members of the semaphorin family have been implicated in mediating axonal guidance in the nervous system by their ability to collapse growth cones and to function as chemorepellents. The present findings show that recombinant Semaphorin D has similar effects on cortical axons and, in addition, inhibits axonal branching. In contrast, semaphorin E acts as an attractive guidance signal for cortical axons. Attractive effects were only observed when growth cones encountered increasing concentrations or a patterned distribution of Semaphorin E, but not when they are exposed to uniform concentrations of this molecule. Specific binding sites for Semaphorin D and Semaphorin E were present on cortical fibers both in vitro and in vivo at the time when corticofugal projections are established. In situ hybridization analysis revealed that the population of cortical neurons used in our experiments express neuropilin-1 and neuropilin-2, which are essential components of receptors for the class III semaphorins. Moreover, semD mRNA was detected in the ventricular zone of the neocortex whereas semE mRNA was restricted to the subventricular zone. Taken together, these results indicate that semaphorins are bifunctional molecules whose effects depend on their spatial distribution. The coordinated expression of different semaphorins, together with their specific activities on cortical axons, suggests that multiple guidance signals contribute to the formation of precise corticofugal pathways.

Animals↗

PubMed1997

Area-specific regulation of gamma-aminobutyric acid type A receptor subtypes by thalamic afferents in developing rat neocortex.

Targeting and innervation of the cerebral cortex by thalamic afferents is a key event in the specification of cortical areas. The molecular targets of thalamic regulation, however, have remained elusive. We now demonstrate that thalamic afferents regulate the expression of gamma-aminobutyric acid type A (GABAA) receptors in developing rat neocortex, leading to the area-specific expression of receptor subtypes in the primary visual (V1) and somatosensory (S1) areas. Most strikingly, the alpha1- and alpha5-GABAA receptors exhibited a reciprocal expression pattern, which precisely reflected the distribution of thalamocortical afferents at postnatal day 7. Following unilateral lesions at the birth of the thalamic nuclei innervating V1 and S1 (lateral geniculate nucleus and ventrobasal complex, respectively), profound changes in subunit expression were detected 1 week later in the deprived cortical territories (layers III-IV of V1 and S1). The expression of the alpha1 subunit was strongly down-regulated in these layers to a level comparable to that in neighboring areas. Conversely, the alpha5 subunit was up-regulated and areal boundaries were no longer discernible in the lesioned hemisphere. Changes similar to the alpha5 subunit were also seen for the alpha2 and alpha3 subunits. These results indicate that the differential expression of GABAA receptor subtypes in developing neocortex is dependent on thalamic innervation, contributing to the emergence of functionally distinct areas.

Animals↗

PubMed1997

Membrane-associated molecules regulate the formation of layer-specific cortical circuits.

The columnar organization of the mammalian neocortex is based on radially oriented axon collaterals which precisely link cells from distinct cortical layers. During development, these interlaminar connections are specific from their initial outgrowth: collaterals form only in the target layers and there are no transient axonal collaterals in the nontarget layers. To examine whether positional cues within individual cortical layers regulate the laminar specificity of collateral formation, explants of cells destined for different cortical layers were cultured on membranes prepared from target and nontarget layers. Axonal growth and branching were examined on homogeneous membrane substrates and on alternating stripes of membranes from different layers. Results show that axons branch preferentially on membrane substrates from those layers that they would target in vivo. In addition, when cortical axons were given a choice to grow on membranes from either their target or their nontarget layer, they exhibited a clear preference for the target layers. This indicates that membrane-associated cues confined to individual layers regulate the formation of collaterals of cortical axons and restrict their growth to their target layers. Heat inactivation of membranes from target layers resulted in reduced axonal branching. The same manipulation of membranes from nontarget layers increased axonal branching for one population of cortical neurons. Taken together, these results suggest that membrane-associated molecules confined to individual layers induce and prevent the formation of axon collaterals in distinct populations of cortical neurons. Thus, the expression of layer-specific cues provides important constraints for the remodeling of local circuits during cortical development.

Animals↗

PubMed1997

How do wiring molecules specify cortical connections?

The laminar and columnar organization of the cortex is reflected in the projections to and from the cortex and in the intracortical connections. During development of the cerebral cortex, growing axons are able to distinguish between the different cortical layers, and cortical axons originating from different laminae respond to different layer-specific signals. Here we consider some experimental systems for identifying mechanisms that contribute to the elaboration of layer-specific cortical connections.

Animals↗

PubMed1997

Tenascin-C synthesis and influence on axonal growth during rat cortical development.

Several putative guidance molecules are restricted to the marginal and subplate zones, the major fibre tracts in the developing cortex. It is presently unknown how their distribution is achieved and how these molecules affect neurite extension. Tenascin-C is of particular interest in this context, because it may either promote or deflect growing axons depending on its mode of presentation. Therefore, the cellular origin of tenascin-C in the developing rat cortex and its effects on the extension of cortical afferents and efferents were examined. Tenascin-C protein is first restricted to the marginal and subplate zones and spreads later into the developing grey matter, in close correlation with afferent innervation. In situ hybridization showed that tenascin-C mRNA is first confined to the ventricular zone, at some distance from the location of the protein, while at later stages tenascin-C-synthesizing cells become scattered throughout the cortical thickness, concomitant with the spread of the protein. In order to assess its function, monoclonal antibodies directed against different domains of tenascin-C were used in a quantitative axonal outgrowth assay. These perturbation experiments suggested that distinct tenascin-C fibronectin type III repeats sustain the growth of thalamic and cortical axons on cortical membrane carpets, whereas the EGF-type repeats are not involved. The combination of different antibodies revealed that separate fibronectin-type III repeats exert cooperative effects. These results suggest that ventricular zone cells regulate the establishment of thalamic and cortical axonal projections through locally restricted deposition of tenascin-C.

Animals↗

PubMed1996

Dual action of a carbohydrate epitope on afferent and efferent axons in cortical development.

During development of the mammalian cerebral cortex, ingrowing afferents from the thalamus take a path that is different from that of axons leaving the cortical plate. Thalamic axons arrive at the cortex at the time before their target cells of layer 4 are generated in the ventricular zone, but they invade the cortex only shortly before these cells have migrated to their final position in the cortex. Growth-promoting molecules are up-regulated in the developing cortical plate during this period. To identify such molecules, we have generated monoclonal antibodies against membrane preparations from rat postnatal cortex. In Western blots, one antibody (mAb 10) recognized a carbohydrate epitope of a glycoprotein with an apparent molecular weight extending from 180 to 370 kDa. Immunohistochemical staining revealed that the staining pattern of mAb 10 at embryonic stages delineates the pathway of thalamocortical axons, with only very faint labeling of the corticofugal pathway. In vitro assays in combination with time-lapse imaging indicated that mAb 10 has opposite effects on the growth of thalamic and cortical axons. The growth speed and axonal elongation of thalamic fibers on postnatal cortical membranes preincubated with mAb 10 was reduced compared with untreated cortical membranes. In contrast, cortical axons grew faster and stopped their growth less frequently after addition of mAb 10 to a cortical membrane substrate. Taken together, these results suggest that a carbohydrate moiety of a membrane-associated glycoprotein plays a role in the segregation of afferent and efferent cortical axons in the white matter. Moreover, the epitope recognized by mAb 10 might also contribute to regulation of the timing of the thalamocortical innervation at later developmental stages.

Aging↗

PubMed1996

Specification of layer-specific connections in the developing cortex.

One of the basic tasks of neurobiology is to understand how the precision and specificity of neuronal connections is achieved during development. In this paper we reviewed some recent in vitro studies on the developing mammalian cerebral cortex that have been made towards this end. The results of these experiments provided evidence that membrane-associated molecules are instrumental for the formation of specific afferent and efferent cortical projections. Substrate-bound molecules guide growing axons towards their target, regulate the timing of thalamocortical innervation and mediate target cell recognition. Moreover, a newly described glycoprotein, defined by a monoclonal antibody, revealed a molecular heterogeneity in the developing white matter. Since this molecule has opposite effects on thalamic and cortical axons, it might play a role in the segregation of axons running to and from the cortex. Substrate-bound cues are important during the formation of local cortical circuits. In vitro assays demonstrated that molecular components confined to individual cortical layers control the laminar specificity of cortical axon branching. This suggests that similar developmental strategies contribute to the laminar specification of extrinsic and intrinsic cortical circuits. Thus substrate-bound molecules might provide the framework for subsequent activity-dependent mechanisms that control the elaboration of precise connections between the cortical columns. A major challenge ahead is to identify the factors that mediate these processes and to determine their mode of action. Recently, two families of proteins, the netrins and the semaphorins/collapsins, have been identified as growth cone signals in the developing spinal cord (reviewed in Goodman, 1994; Colamarino and Tessier-Lavigne, 1995a; Dodd and Schuchardt, 1995; Kennedy and Tessier-Lavigne, 1995). Semaphorins/collapsins appear to regulate axonal guidance by repelling growth cones and by inhibiting axonal branching and synapse formation. Originally, netrins have been purified as diffusible chemoattractants for commissural axons of the dorsal spinal cord, but it is now well established that they can also function as chemorepellent factors for other classes of neurons. Since netrins are related to extracellular matrix components and since they can bind to the cell surface, they might also act as local guidance cues. A possible role of netrins and semaphorins/collapsins in the development of cortical connections is likely to be resolved in the near future. The identification of the factors that regulate specific branching patterns of cortical neurons might provide a better understanding of cortical development, but it might also be relevant to some aspects of plasticity and repair in the adult cortex.

Afferent Pathways↗

PubMed1996

Developmental strategies underlying the elaboration of cortical circuits.

The mammalian cerebral cortex is organized in layers and columns, which are reflected in the local intrinsic connections and in the projections to and from the cortex. It is well established that the development of the columnar architecture is under the influence of neuronal activity, but little is known about the mechanisms that control the laminar specificity of cortical circuits. Here we review some recent studies which show that diffusible and membrane-associated molecules provide sufficient information to reconstruct layer-specific intrinsic and extrinsic cortical circuits under in vitro conditions.

Animals↗

PubMed1995

Guidance of thalamocortical axons by growth-promoting molecules in developing rat cerebral cortex.

Substrate-bound guidance cues play an important role during the development of thalamocortical projections. We used time-lapse video microscopy to study the growth behaviour of thalamic axons on different substrates. On embryonic cortical membranes and on a pure laminin substrate, thalamic fibres advanced relatively slowly (approximately 15 microns/h) and on average their growth cones retracted transiently every approximately 5 h. In contrast, on membranes prepared from early postnatal cortex, thalamic fibres grew twice as fast and spontaneous growth cone collapse occurred approximately 8 times less often. Experiments in which we used the sugar-binding lectin peanut agglutinin or heat inactivation to change the membrane properties indicated that these differences are due to growth-supporting molecules on postnatal cortical membranes. When offered a choice between embryonic and postnatal cortical membranes, thalamic axons preferred the postnatal membrane substrate. Time-lapse imaging revealed that borders between these two substrates effectively guided thalamic fibres, and in most cases axons changed their direction without collapse of the growth cone. Our results suggest that thalamic axons can be guided by the spatial distribution of growth-promoting molecules in the developing cortex.

Animals↗

PubMed1995

The specification of neuronal fate: a common precursor for neurotransmitter subtypes in the rat cerebral cortex in vitro.

Neurotransmitter choice is a crucial step in neural development. In the cerebral cortex, pyramidal neurons use the excitatory neurotransmitter glutamate, whereas non-pyramidal cells use the inhibitory neurotransmitter GABA. We are interested in how these two neuronal types are generated. We labelled precursor cells from embryonic rat cerebral cortex with a retroviral vector in dissociated cell cultures, and examined the neurotransmitter phenotype of their progeny immunohistochemically after 2 weeks in vitro. We discovered, first, that precursor cells in culture generate glutamatergic and GABAergic neurons in proportions similar to those in vivo. Second, we found that neuronal precursor cells gave rise to both GABAergic and glutamatergic neurons. These results suggest that neuronal precursor cells in the cerebral cortex have the potential to generate both neuronal subtypes. Moreover, these data are consistent with a stochastic model of neurotransmitter specification.

Animals↗

PubMed1995

The specificity of interactions between the cortex and the thalamus.

The functioning of the adult mammalian cerebral cortex depends critically upon precise interconnections between specific thalamic nuclei and distinct cortical regions. Therefore, one central issue in understanding cortical development is determining the cellular and molecular strategies underlying the specification of thalamocortical projections. We address the role of axon-axon interactions and membrane-bound guidance molecules in the establishment of the development of layer-specific patterns of afferent and efferent cortical connections does not depend upon neuronal activity. We present evidence that activity conveyed by thalamic afferents is required for the elaboration of the columnar specificity of cortical circuits.

Animals↗

PubMed1995

Relationships between dendritic fields and functional architecture in striate cortex of normal and visually deprived cats.

We examined relationships between the pattern of geniculocortical innervation and the dendritic fields of cells in layer 4 of in cat primary visual cortex. Experiments were performed on normal animals and on cats in which the geniculocortical projection was altered by monocular deprivation or by the induction of divergent squint during the critical period. Thalamic afferents providing the input from the contralateral eye were anterogradely labeled by injecting the fluorescent tracer Dil into lamina A of the lateral geniculate nucleus. Intracellular staining with Lucifer yellow in slice preparations allowed simultaneous visualization of the morphology of individual cells and the thalamic afferents. Our results demonstrate that spiny stellate cells close to the upper and lower margin of the geniculocortical input have highly asymmetric dendritic fields, and thereby confine their dendrites to the termination zone of these afferents. This effect was specific for the cell class; it was not observed in pyramidal neurons. These dendritic asymmetries perpendicular to the laminar borders of spiny stellate cells were not altered by monocular deprivation or strabismus. In contrast, visual deprivation strongly influenced the dendritic arbors of spiny stellate cells near the borders between adjacent ocular dominance columns. In normal animals, the dendrites of cells near columnar borders remained preferentially within one column. These dendritic asymmetries became much more pronounced in strabismic animals. Monocular deprivation weakened the influence of the columnar borders on dendritic fields. Spiny stellate cells within the columns of the open eye exhibited a slight tendency to confine their dendrites to these columns. Cells in the columns of the deprived eye showed the opposite effect; they extended their dendrites preferentially into the adjacent columns of the open eye. These results demonstrate that the segregation of geniculocortical afferents into ocular dominance columns and its perturbation by manipulation of the visual input plays an important role in defining the morphology of cortical target cells. Thus, activity-dependent structural changes not only occur at the level of the presynaptic terminals, but also at the level of the postsynaptic target cells, and thereby contribute to build up the functional architecture of the cortex.

Animals↗

PubMed1994

GABAA receptor alpha 1 subunit, an early marker for area specification in developing rat cerebral cortex.

Changes in the expression of neurotransmitter receptors in developing cerebral cortex may be related to the functional maturation of distinct areas. In the present study, we have tested whether GABAA receptor expression in neonatal rats reflects the differentiation of cortical areas. Specifically, the alpha 1 subunit, one of the most prevalent GABAA receptor subunits in adult cerebral cortex, is up-regulated postnatally, suggesting a link with the establishment of inhibitory circuits. Using immunohistochemistry with a subunit-specific antiserum, we observed a striking area- and lamina-specific increase in staining for GABAA receptors containing the alpha 1 subunit (alpha 1-GABAA receptors), from low levels in neonates to an intense and uniform staining in adults. Already at birth, the alpha 1-subunit immunoreactivity selectively demarcated the boundaries of certain cortical areas. In particular, the primary somatosensory (S1) and visual (V1) areas were distinctly delineated with a band of alpha 1-subunit immunoreactivity located in the developing layers III and IV. The staining ended abruptly at the presumptive boundaries of S1 and V1, adjacent areas being unstained at this age. Around postnatal day 3, clusters of alpha 1-subunit positive cells were seen in layers III-IV of S1 and V1 extending their dendrites up to layer I, where they arborized profusely. In addition, the distribution of alpha 1-GABAA receptors in S1 revealed in detail the differentiation of the barrel field during early postnatal development. Although staining was observed in all areas by postnatal day 6, differences in the laminar distribution of alpha 1-GABAA receptors persisted for at least 1 more week. Our results provide evidence for the existence of area-specific boundaries in neocortex of newborn rats before layers III-IV are fully differentiated and innervated by cortical afferents. Furthermore, the area- and lamina-specific maturation of alpha 1-GABAA receptor staining demonstrates the value of this marker for investigating the cytoarchitectonic differentiation of cortical areas during development.

Animals↗

PubMed1994

Differentiation of transmitter phenotypes in rat cerebral cortex.

Cortical neurons differ in their neurochemical properties. Projection neurons use excitatory amino acids as transmitters, most local interneurons contain the inhibitory transmitter GABA, and specific subtypes of local circuit neurons express distinct neuropeptides. How this cellular diversity is generated during development is not known. We have been studying the transmitter differentiation of cortical neurons in different in vitro systems using immunohistochemical techniques. Transmitter phenotypes of cortical neurons were examined in slice cultures, i.e. in the absence of extrinsic cortical connections, and in dissociated cortical cell cultures, i.e. in the absence of extrinsic and intrinsic cortical connections. The expression of vasoactive intestinal polypeptide in cortical interneurons occurred normally in slice cultures prepared from neonatal rats between birth and 2 days of age, but was strongly impaired in dissociated cell cultures prepared at the same time. These results suggest that the intact cortical environment present in the slice cultures exerts crucial influences for neuropeptide differentiation. In contrast, the transmitters glutamate and GABA were expressed normally in the appropriate cell types and similar in proportions in dissociated cell cultures prepared from cortices at embryonic day 19. Only cells dissociated during S-phase failed to express glutamate and GABA in vitro. When cells were kept for 24 h after mitosis in a cortical slice preparation in vitro, however, they later expressed their appropriate transmitter phenotypes. Thus, signals from the local cortical environment that act early in the cell cycle are required for the specification of transmitter phenotypes of cortical neurons.

Animals↗